Abstract

Obesity induced by Western diets is associated with type 2 diabetes mellitus and cardiovascular diseases, although underlying mechanisms are unclear. We investigated a murine model of diet-induced obesity to determine the effect of transient potential receptor vanilloid 1 (TRPV1) deletion on hypertension and metabolic syndrome. Wild-type and TRPV1 knockout mice were fed normal or high-fat diet from 3 to 15 weeks. High-fat diet-fed mice from both genotypes became obese, with similar increases in body and adipose tissue weights. High-fat diet-fed TRPV1 knockout mice showed significantly improved handling of glucose compared with high-fat diet-fed wild-type mice. Hypertension, vascular hypertrophy, and altered nociception were observed in high-fat diet-fed wild-type but not high-fat diet-fed TRPV1 knockout mice. Wild-type, but not high-fat diet-fed TRPV1 knockout, mice demonstrated remodeling in terms of aortic vascular hypertrophy and increased heart and kidney weight, although resistance vessel responses were similar in each. Moreover, the wild-type mice had significantly increased plasma levels of leptin, interleukin 10 and interleukin 1β, whereas samples from TRPV1 knockout mice did not show significant increases. Our results do not support the concept that TRPV1 plays a major role in influencing weight gain. However, we identified a role of TRPV1 in the deleterious effects observed with high-fat feeding in terms of inducing hypertension, impairing thermal nociception sensitivity, and reducing glucose tolerance. The observation of raised levels of adipokines in wild-type but not TRPV1 knockout mice is in keeping with TRPV1 involvement in stimulating the proinflammatory network that is central to obesity-induced hypertension and sensory neuronal dysfunction.

Introduction

Transient receptor potential type 1 (TRPV1) is a nonselective ion channel, primarily localized to C- and Aδ-fiber sensory nerves.1 These fibres have a dual sensory-efferent role, involving nociception and release of vasodilator neuropeptides, such as calcitonin gene-related peptide.2 A range of endogenous activating mechanisms are known.1,3 Capsaicin is used as an experimental tool, as an agonist, and also as its continued application desensitizes and defunctionalizes sensory nerves.1 TRPV1 knockout (KO) mice possess a phenotype of reduced responsiveness to thermal thresholds,4,5 a lack of vasodilatation to capsaicin,6 and decreased joint inflammation.7 TRPV1 deletion is associated with proinflammatory consequences in cardiovascular alterations, such as the ones observed in sepsis,8 myocardial ischemia,9,10 and DOCA-salt–induced hypertension.11 We have demonstrated both anti- and proinflammatory effects of TRPV1 deletion6,8 in support of the concept that TRPV1 can act as a molecular integrator and can play pivotal roles in disease progression.

TRPV1 channel activation and high calcium influx were originally shown to prevent adipogenesis,12 but not more recently.13 In vivo studies involving a capsaicin-containing diet, that on a chronic basis desensitizes sensory nerves in addition to the TRPV1 channel, are associated with a leaner phenotype.12 Moreover, wild-type (WT) mice fed a 11% high-fat diet (HFD) for 3 to 25 weeks gained more weight than TRPV1KO mice, but only during the 15- to 25-week period, suggesting a link with age-onset obesity only. In contrast, capsaicin-pretreatment of Zucker rats (a model of the metabolic syndrome and type 2 diabetes mellitus) led to a greater increase in weight after 12 weeks of capsaicin feeding, with the capsaicin-fed rats protected against the normal deterioration of glucose handling.14 Also, HFD-TRPV1KO mice exhibited improved glucose tolerance compared with HFD-WT mice.15 Therefore, a complex picture has emerged of the role of TRPV1, with interest in the concept that TRPV1 may play a primary role in body weight regulation and metabolism.16

Based on evidence that TRPV1 may influence obesity and glucose handling in a harmful manner, we have investigated the influence of TRPV1 in a model involving WT and TRPV1KO mice. Our results show that HFD-induced hypertension is not observed when TRPV1 is deleted and that HFD-WT mice become less responsive to the characteristic TRPV1-dependent thermal nociception.

Materials and Methods

An expanded Methods section is available in the online-only Data Supplement.

Mouse Models

C57BL6/129SVJ TRPV1KO mice and their genetically unaltered WT counterparts were bred and characterized.7,8 Mice were divided into sex-balanced groups and fed an HFD (35% fat from lard) or normal diet (ND; see the online-only Data Supplement for details), for 12 weeks from 3 weeks of age, according to the Animals (Scientific Procedures) Act, 1986, United Kingdom.

Blood Pressure and Additional Analysis

Blood pressure was assessed in restrained conscious mice using the tail-cuff CODA plethysmography technique17 and by a preimplanted telemetry device (PA-C10, DSI) in unrestrained conscious mice. At 15 weeks of age, after blood pressure measurements were complete, blood glucose was measured before (baseline) and after glucose administration (2 mg/kg IP).18 Nociception assays are detailed in the online-only Data Supplement. At termination, external markers of obesity and development were taken and samples were collected for analysis.

Statistical Analysis

Results are shown as mean±SEM and evaluated by 2-way ANOVA and Bonferroni posttest, unless stated. Significance was accepted at P≤0.05.

Results

Effect of HFD on Body Weight

Mice on the HFD gained significantly more weight than ND mice. Weight gain was similar in HFD-WT and HFD-TRPV1KO mice (Figure 1A). However, assessment of adipocyte size revealed that HFD-TRPV1KO mesenteric adipose tissue contained a significantly higher percentage of small adipocytes (1–20 µm) than that from HFD-WT mice (Figure 1B). Weight gain was paralleled by a significant increase in abdominal circumference (Table S1) and fat mass (Figure S1). Typically, mice ate less mass, despite increased calorific intake, over the first 28 days of feeding in the HFD group, irrespective of genotype (Figure 1C). Circulating levels of leptin increased in both HFD-WT and HFD-TRPV1KO mice (Figure S1). Adiponectin levels were significantly increased in HFD-WT but not HFD-TRPV1KO mice (Figure S1).

Effect on Glucose Tolerance

Baseline glucose levels were similar in WT and TRPV1KO mice but showed a nonsignificant raised trend in HFD mice (Figure 4A). Glucose administration in HFD-WT and HFD-TRPV1KO mice caused substantial increase in plasma glucose levels compared with ND mice. However, by comparison, after the administration of the glucose load, the HFD-TRPV1KO mice were able to reverse the high plasma levels quicker than the HFD-WT group (Figure 4B), with a significant difference when areas under the response curve (0–105 minutes) were compared for WT-HFD and WT-TRPV1KO mice (Figure 4C). In addition, the HFD-WT and HFD-TRPV1KO mice exhibited increased heart size and heart rate (Figure S6) when compared with ND mice. By comparison, kidney weight in HFD-WT mice was significantly increased compared with TRPV1KO mice (Figure 4D), although not when calculated as a kidney:body weight ratio (Figure S7), where no structural damage was observed (Figure S7).

Effect on Nociceptive Thresholds

The characteristic phenotype of TRPV1KO mice is that they are insensitive to thermal nociceptive stimuli.7 Little is known about the impact of HFD on TRPV1-dependent heat sensitivity. Here, HFD-WT mice showed increased latency to heat when compared with ND-WT mice, indicating a loss in heat sensitivity in the skin of the HFD-WT mice (Figure S8). Both HFD-WT and HFD-TRPV1KO mice exhibited similar and reduced sensitivity to mechanical hyperalgesia when compared with their ND controls (Figure S8).

Effect on Plasma Protein Levels

Because obesity is associated with a low-grade inflammation, and inflammation is also a key event in cardiovascular disease, we determined levels of circulating cytokines in a subset of the mice. Plasma levels of interleukin (IL) 10 and IL-1β were significantly raised in HFD-WT but not HFD-TRPV1KO mice compared with normally fed mice. In addition, IL-10 and IL-1β levels were significantly higher in the HFD-WT when compared with the HFD-TRPV1KO group. There was evidence of raised levels (nonsignificant) of IL-6 and IL-12 in HFD-WT mice only (Figure 5). Tumor necrosis factor-α and interferon-γ levels were not raised beyond the baseline levels (Figure S8). The possibility of ongoing vascular inflammation was examined through measurement of circulating endothelin 1, but no significant increase in its levels were observed (Figure S9).

Discussion

This study provides novel evidence of a linked series of events whereby TRPV1 plays a primary and pivotal role in the development of the metabolic syndrome. The HFD-WT mice show an enhanced metabolic syndrome compared with the HFD-TRPV1KO mice, in terms of hypertension, glucose handling, and low-grade inflammation, despite similar weight gain. The results show, for the first time to our knowledge, that TRPV1 deletion is associated with protection against obesity-induced hypertension and inhibition of low-grade inflammation. In addition, the HFD-WT mice show an altered nociceptive phenotype with loss of sensitivity to heat touch that is at least partly dependent on TRPV1. The results are in keeping with the concept that a series of complex networks operate in obesity-driven dysfunction that leads to hypertension and that TRPV1 is an influential component in this network.

The HFD model was based on previous studies where obesity-induced hypertension was defined in C57BL/6 mice.19–21 The results confirm that WT and TRPV1KO mice gain similar weight when fed an HFD from 3 to 15 weeks.13 Studies of TRPV1 in weight loss present mixed results12,14,22 and have often involved chronic administration of capsaicin, which causes the destruction and defunctionalization of the sensory nerves, in addition to the TRPV1 channel. Interestingly, a novel capsaicin analogue has been shown to be well tolerated but to have no effect on body weight in humans, despite significantly reducing abdominal fat in a manner that was linked with 2 common genetic variants.23 Our results, when taken with those already in the literature, indicate that TRPV1 is unlikely to play a major role in influencing weight gain. Of note, we show that HFD-WT mice possess a greater percentage of larger adipocytes in keeping with the concept that functional TRPV1 is implicated in normal preadipocyte-to-adipocyte differentiation.12 We confirm that lack of active TRPV1 is able to attenuate loss of glucose handling, as shown previously in HFD-TRPV1KO17 and TRPV1 antagonist-treated mice.18

Blood pressure was measured by tail cuff or telemetry in 3 separate experiments. All of the experiments showed that HFD-TRPV1KO mice were protected from HFD-induced hypertension. TRPV1 activation releases vasodilator neuropeptides, leading to peripheral vasodilatation.5 We originally hypothesized that TRPV1 deletion would trigger a loss of the vasodilator effect and hypertension, but this is not the case with TRPV1 presence associated with increased blood pressure in this HFD model. The obesity-induced high blood pressure in HFD-WT but not HFD-TRPV1KO mice was mirrored by an effect on vascular hypertrophy in HFD-WT mice, indicating that the onset of vascular remodeling and these 2 phenomenon may well be directly related. Further studies using antihypertensives would verify this. However, vascular inflammation, as assessed by vascular cell adhesion protein 1 immunohistochemistry, was not increased in either HFD-WT or HFD-TRPVIKO aortas. Vascular cell adhesion protein 1 is often upregulated in the aorta of the hypertensive mouse, as shown by this laboratory in the angiotensin II hypertension model.17 Myograph studies of mesenteric resistance vessels were carried out. Our results revealed that constrictor and dilator responses to phenylephrine, calcitonin gene-related peptide, and the endothelial-dependent carbachol remained intact, suggesting little vascular dysfunction at the mesenteric resistance vessel level at least. It has been shown recently that dietary capsaicin resists loss of endothelial-dependent relaxation and hypertension in HFD-fed mice,24 but whether this is associated with activating or desensitizing doses of capsaicin has been debated.25

We observed an increased kidney weight in HFD-WT but not HFD-TRPV1KO mice, but not when calculated as a kidney:body weight ratio (Figure S14). This was not associated with structural damage, as shown by histology. Thus, no obvious link between kidney damage and hypertension was observed. Moreover, a similar increase in heart rate and weight gain was observed in HFD mice, irrespective of genotype, indicating that the heart, in our model, is influenced by the HFD in a similar manner, independent of genotype.

TRPV1 deletion enhances damage after myocardial infarction, mainly because of a lack of neuropeptide release.12,14 Obesity and the onset of the metabolic syndrome are associated with an altered phenotype in terms of inflammatory markers and the presence of a low-grade inflammation,26 although mechanisms by which this translates into a systemic condition are unclear. Here, we found raised levels of leptin in HFD-WT and HFD-TRPV1KO mice, reaching significance in the HFD-WT mice, indicating that adipose tissue is involved at a primary level. Furthermore, levels of adiponectin and IL-10 were raised, both suggested to be anti-inflammatory in hypertension, although their levels have been shown to be high in patients with chronic heart failure.27 The lack of detection of increased levels of IL-10 in HFD-TRPV1KO mice suggests that TRPV1 does not play a central role in influencing a shift from the T-helper 1 to a T-helper 2 cytokine profile. We found evidence for a range of circulating cytokines that included IL-1β, IL-6, and IL-12 in HFD-WT but not HFD-TRPV1KO mice. Of these levels, IL-1β reached significance, and we assume that the high levels of IL-10 acted to decrease the expression and release of proinflammatory cytokines, in keeping with its established activity. We did not detect increased circulating levels of tumor necrosis factor-α, which is considered to function as an autocrine/paracrine factor in adipose tissue, rather than a circulating factor, and these results are in keeping with that concept.28

The inflammatory cytokine profile fit with the dysfunction of glucose handling and the hypertensive profile, rather than directly with adiposity. Indeed, it is tempting to link circulating levels of the cytokines with the observed increase in blood pressure, but the understanding of the exact role of the cytokines is at an early stage. Endogenous IL-10 has been suggested to limit the onset of angiotensin II–mediated damage29 and may be acting here to suppress inflammatory events at the vascular wall. We examined the possibility that vascular inflammation may be increased in HFD-WT but not HFD-TRPV1KO mice by measuring endothelin 1 levels. Endothelin 1 levels are suggested to become functionally significant at an early stage in the spontaneously hypertensive rats and to be linked with insulin resistance.30 The vasodilator potential of TRPV1 activation is suppressed in pigs with metabolic syndrome.31 A recent study has revealed a mechanism by which the vasoconstrictor peptide endothelin is released to mediate pressor responses in WT but not TRPV1KO mice after TRPV1 activation by capsaicin.32 However, increased circulating levels of endothelin could not be detected in this study. Both IL-1β and IL-6 have also been linked to hypertension and, in the case of IL-6, T-cell activation and insulin resistance.33,34 These collected findings suggest a need to learn more about the interplay between the different facets of the metabolic syndrome and the factors that lead to the onset of hypertension.

Finally, TRPV1KO mice typically demonstrate a slowed response in terms of paw withdrawal threshold to heat.7 A reduced response to thermal thresholds has been demonstrated in Zucker rats.35 Here, concomitant analysis of 2 distinct TRPV1-dependent pathways in the same mouse indicates that TRPV1-dependent sensitivity to heat is altered during the same time frame as the vascular changes occur, before the establishment of defined type II diabetes mellitus. We also demonstrate a loss of mechanical sensitivity that is TRPV1 independent. Thus, of note, changes in nociception sensitivity that are presently associated with diabetes mellitus occur at an early time point in this model that is more relevant to the metabolic syndrome.

In conclusion, we confirm TRPV1 deletion has no effect on the onset of weight gain in mice, even in the presence of an HFD, over 8 to 15 weeks. On the other hand, we provide evidence that TRPV1KO mice were protected from obesity-induced hypertension, low-grade inflammation, and glucose tolerance.

Perspectives

The results demonstrate for the first time a key influence of TRPV1 in the onset of obesity-induced hypertension, low-grade inflammation, and a related reduction in thermal sensation. These findings suggest that TRPV1 may play a critical role in the onset of symptoms of the metabolic syndrome. These changes are observed before any effect that TRPV1 may have in influencing weight gain. These results complement very recent findings that a TRPV1 antagonist protects against the onset of glucose tolerance in a murine model of type II diabetes mellitus. However, it is important to note that these results contrast with studies that have concentrated previously on cardiovascular disease (primarily ischemic conditions) where lack of TRPV1 is associated with a worsened phenotype. These studies provide further evidence of a critical regulatory role of TRPV1 in metabolic and cardiovascular regulation. They indicate a possible novel therapeutic approach whereby TRPV1 antagonists may protect against onset of the metabolic syndrome.

Acknowledgments

We acknowledge the support of Chi Teng Vong and Khadija Alawi.

Sources of Funding

This work was supported by the British Heart Foundation (to Dr Marshall); a Capacity Building Award in Integrative Mammalian Biology funded by the British Biotechnology Science Research Council, British Pharmacological Society, Higher Education Funding Council, Knowledge Transfer Network, Medical Research Council, and Scottish Funding Council (to Drs Liang and Brain); Arthritis Research United Kingdom (to Dr Fernandes; grant No. 19296); British Biotechnology Science Research Council (to Dr Smillie); and Diabetes United Kingdom (to Dr Dessapt-Baradez).